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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 - 31 May 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study has been performed in accordance with OECD 209 (2010) and according to GLP principles. Deficiencies: in the limit/range-finding test the oxygen uptake rate in the blank control was slightly below the guideline requirement (20 mg oxygen per one gram of activated sludge) and nitrification inhibition has not been fully investigated.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(2010)
Deviations:
yes
Remarks:
in the limit/range-finding test the oxygen uptake rate in the blank control was slightly below the guideline requirement (20 mg oxygen per one gram of activated sludge) and nitrification inhibition has not been fully investigated.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 mL of test substance mixtures in tap water purified by reverse osmosis with initial loading rates of 2.5 times the final loading rate (1000 mg/L) . These mixtures were stirred in closed dark brown bottles for approximately 25 hours. In the limit test pH of the loading rates prepared at 2500 mg/L was adjusted from 2.3, 2.4 and 2.3 to 5.2, 8.1 and 7.0 respectively, using 1N NaOH.
- Controls: Test medium without test substance but treated in the same way as the test substance solutions
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not reported
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Method of cultivation: The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for both tests). The pH was 7.1 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
during which aeration and stirring took place
Hardness:
Not reported
Test temperature:
- Combined limit/range-finding test: 18.2 - 20.2 °C
- Limit test: 19.4 - 21.9 °C
pH:
-Combined limit/range-finding test
Before addition of sludge: 7.4 - 7.5 (Blank control); 2.9 - 7.2 (test solutions)
After 3h contact time: 8.1 - 8.3 (Blank control); 4.2 - 8.3 (test solutions)

- Limit test
Before addition of sludge: 7.4 - 7.8
After 3h contact time: 7.5 - 7.8
Nominal and measured concentrations:
- Nominal
Combined limit/range-finding test): blank control, 10, 100 and 1000 mg/L (loading rate)
Limit test: blank control and 1000 mg/L (loading rate)
Details on test conditions:
TEST SYSTEM (Combined limit/range-finding test)
- Test vessel: All glass, 1 litre test bottles
- Aeration: Clean, oil-free air
- No. of vessels per concentration (replicates): 3 for the highest test concentration (100 mg/L loading rate) and 1 for the lowest test concentrations (10 and 100 mg/L loading rates)
- No. of vessels per control (replicates): 6 for the blank control; 1 for the nitrification control; 1 for the abiotic control, 1 for the toxicity control
- Biomass loading rate (suspended solids): 1.5 g/L

TEST SYSTEM (Limit test)
- Test vessel: All glass, 1 litre test bottles
- Aeration: Clean, oil-free air
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2 (blank control)
- Biomass loading rate (suspended solids): 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH and temperature determined before the addition of sludge and at the end of the exposure period

TEST DESIGN
- Performance of the test: The synthetic medium (16 mL) and an appropriate amount of the test substance loading were mixed and made up to 250 mL with tap-water purified by reverse osmosis in a 1 litre bottle. The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device.
- Aeration: adjusted to obtain a dissolved oxygen concentration at the start of the test above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- Note: The limit test was a standard test that did not include a nitrification inhibitor or abiotic conditions.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes (limit test)
- Photoperiod: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Oxygen consumption (respiration rate). After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 9-17 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. Determination of oxygen was performed with oxygen probes connected to a BlueBox, a multichannel measuring and controlling system.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: At neutralized pH
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: At neutralized pH
Details on results:
- Combined limit/range-finding test: No inhibition was observed at a loading rate of 10 and 100 mg/L. An average of 99% inhibition of the respiration rate was observed at a loading rate of 1000 mg/L. However, the very low pH at a loading rate of 1000 mg/L might have caused and/or increased the inhibition of the microbial activity. Therefore the test was repeated at the highest test concentration (1000 mg/L loading rate) with the pH adjusted to 7.4 - 7.6. There was no oxygen uptake from abiotic processes. The results at 1000 mg/L with a nitrification inhibitor showed 95% heterotrophic inhibition of the respiration rate.

- Limit test: No inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg/L at neutralized pH. Thus the toxic effect observed at a loading rate of 1000 mg/L in the combined limit/range-finding test was caused by the low pH and not the intrinsic toxicity of FDCA.

- Acceptability of the test:
1. In the limit test the mean control oxygen uptake rate was 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. In the combined limit/range-finding test the mean control oxygen uptake rate was just below 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (18 mg O2/g.h). The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the limit test.
2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (13 and 9 mg/L in the combined limit/range-finding and limit test respectively)
Results with reference substance (positive control):
3,5-dichlorophenol (final test): 9.4 mg/L
Validity criteria fulfilled:
yes
Conclusions:
FDCA at neutralized pH was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/L (NOEC). The EC50 was at a loading rate of >1000 mg/L.
Executive summary:

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209, microorganisms in activated sludge were exposed to FDCA at the following nominal concentrations (limit test): 0 (blank control) and 1000 mg/L (loading rate). The pH of the test solution was adjusted before the start of test. Test substance at neutralized pH was not toxic to waste water (activated sludge) bacteria at an exposure concentration of 1000 mg/L (NOEC). The EC50 was >1000 mg/L based on nominal concentrations. In the limit/range-finding test the oxygen uptake rate in the blank control was slightly below the guideline requirement (20 mg oxygen per one gram of activated sludge) and nitrification inhibition has not been fully investigated. Therefore, the study is considered to be reliable with restrictions.

Description of key information

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209, microorganisms in activated sludge were exposed to FDCA at the following nominal concentrations (limit test): 0 (blank control) and 1000 mg/L (loading rate). The EC50 was >1000 mg/L based on nominal concentrations. In the limit/range-finding test the oxygen uptake rate in the blank control was slightly below the guideline requirement (20 mg oxygen per one gram of activated sludge) and nitrification inhibition has not been fully investigated. Therefore, the study is considered to be reliable with restrictions.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

In a 3 h activated sludge respiration inhibition study conducted according to OECD guideline 209, microorganisms in activated sludge were exposed to FDCA at the following nominal concentrations (limit test): 0 (blank control) and 1000 mg/L (loading rate). The pH of the test solution was adjusted before the start of test. Test substance at neutralized pH was not toxic to waste water (activated sludge) bacteria at an exposure concentration of 1000 mg/L (NOEC). The EC50 was >1000 mg/L based on nominal concentrations. In the limit/range-finding test the oxygen uptake rate in the blank control was slightly below the guideline requirement (20 mg oxygen per one gram of activated sludge) and nitrification inhibition has not been fully investigated. Therefore, the study is considered to be reliable with restrictions.